Higher plants produce a variety of secondary metabolic compounds, such as alkaloids, which are useful in the treatment of human diseases. One recently discovered alkaloid group, the taxanes, includes taxol, a diterpenoid sesquiterpene that exhibits strong antineoplastic activity and shows great promise in the treatment of leukemia, melanoma, breast cancer and ovarian cancer. (Brossi, Einsig, Rowinski, Wani).
Taxol was originally isolated from the bark and leaves of the Pacific Yew, Taxus brevifolia Nuttall, where it is present in minute quantities, generally less than 0.015% by weight of tissue extracted (Vidensk). Taxol has been isolated from other species of Taxus such as T. cuspidata, T. baccata, and T. media. The amount of taxol obtainable from different Taxus species is variable (Vidensk, Wani). This source of taxol is quite limited, due to the large number of trees which must be harvested to obtain therapeutic amounts of the compound. It has been estimated that dozens of trees may be required to obtain a therapeutic dose for a single patient. Unfortunately the best natural plant source of taxol is T. brevifolia, a slow-growing tree restricted to a narrow geographical range in old growth, mixed-conifer forest in the Pacific Northwest.
Synthetic production of taxol has proved impractical, due to the complexity of the alkaloid synthesis. Semi-synthetic methods of taxol production, in which a taxol precursor is converted to taxol synthetically has also been proposed (U.S. Pat. No. 5,015,744). The semi-synthetic method is also impractical at present, due to the difficulty in obtaining purified precursors.
It has been suggested that the limitations in taxol production might be overcome through the use of tissue culture techniques to produce secondary metabolites, including taxol. Success, however, requires first obtaining cells which are capable of sustained growth in culture, and secondly, inducing such cells to produce adequate quantities of the desired metabolite. In particular, the production of significant amounts of secondary metabolites in tissue culture has met with numerous difficulties. (Collinge, Dicosmo, Evans; see also U.S. Pat. No. 4,910,138 to Miura et al., column 1, lines 18-35)
One source of difficulty is that cultured cells often fail to synthesize the desired metabolite in culture. Although callus tissue may contain some concentration of secondary metabolites at the time of callus induction, subsequent subcultures of the callus display progressively lower concentrations of secondary metabolites. In such cases, tissue culture production of secondary metabolites can be ineffective because it requires continual harvest of explants from differentiated plant bodies. Secondary metabolite production is further limited because production is often contingent on cell differentiation or organization of the cells into organized tissues.
Production of sporophyte-derived callus tissue and extraction of taxol therefrom has been reported for T. brevifolia (U.S. Pat. No. 5,019,804 to Christen et al.). The method disclosed in the '504 patent for production of taxol from culture follows the procedure disclosed earlier by Misawa et al. (Misawa, 1982, 1983) for the production of a variety of antineoplastic drugs from plant tissue cultures, including taxol from T. brevifolia. The method involves inducing callus from explants of the selected plant source, e.g., T. brevifolia, growing the callus cells in a cell suspension, and isolating the desired secondary metabolite antineoplastic agent, e.g., taxol, from the suspension culture. In studies conducted in support of the present invention, the approach generally disclosed in the '504 has yielded levels of taxane which are in the range of a few parts per billion fresh tissue weight.